US20190240298A1 - Methods for treating farber disease - Google Patents

Methods for treating farber disease Download PDF

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US20190240298A1
US20190240298A1 US16/263,700 US201916263700A US2019240298A1 US 20190240298 A1 US20190240298 A1 US 20190240298A1 US 201916263700 A US201916263700 A US 201916263700A US 2019240298 A1 US2019240298 A1 US 2019240298A1
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rhac
dose
aliquot
group
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Eric Gaukel
Brante SAMPEY
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Enzyvant Farber F/k/a Enzyvant Sciences GmbH GmbH
Enzyvant Therapeutics GmbH
Aceragen Inc
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Enzyvant Farber GmbH
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Assigned to ENZYVANT FARBER GMBH reassignment ENZYVANT FARBER GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GAUKEL, Eric, SAMPEY, Brante
Assigned to ENZYVANT THERAPEUTICS GMBH reassignment ENZYVANT THERAPEUTICS GMBH MERGER AND CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: ENZYVANT FARBER GMBH, ENZYVANT THERAPEUTICS GMBH
Assigned to ENZYVANT FARBER GMBH, F/K/A ENZYVANT SCIENCES GMBH reassignment ENZYVANT FARBER GMBH, F/K/A ENZYVANT SCIENCES GMBH NUNC PRO TUNC ASSIGNMENT (SEE DOCUMENT FOR DETAILS). Assignors: ROIVANT SCIENCES, INC.
Assigned to ROIVANT SCIENCES, INC. reassignment ROIVANT SCIENCES, INC. NUNC PRO TUNC ASSIGNMENT (SEE DOCUMENT FOR DETAILS). Assignors: GAUKEL, Eric, SAMPEY, Brante
Assigned to ACERAGEN, INC. reassignment ACERAGEN, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: ENZYVANT THERAPEUTICS GMBH
Assigned to NOVAQUEST CO-INVESTMENT FUND XV, L.P. reassignment NOVAQUEST CO-INVESTMENT FUND XV, L.P. SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ACERAGEN, INC.
Priority to US17/736,690 priority patent/US20230123505A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01045—Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase

Definitions

  • Farber disease a lysosomal storage disorder (LSD) is a condition that was first described in 1952 in a 14-month-old infant with granulomatous lesions on multiple joints and evidence of lipid storage. Over the ensuing decade other similar cases were described, all of which demonstrated similar lesions and often exhibited a characteristic “hoarse” cry or voice due to the presence of lesions on the larynx. The involvement of other organ systems in some of these patients, including the lung, liver, spleen and central nervous system (CNS), also was noted.
  • CNS central nervous system
  • HSCT Hematopoietic stem cell transplantation
  • HED human equivalent dose
  • FIG. 3 shows that the juvenile CD-1 mouse rhAC (RVT-801) pharmacokinetic profile is similar to the Farber mouse experimental rhAC activity profile in circulation. This indicates that the juvenile CD-1 mouse provides an appropriate approximation of the Farber mouse PK, and as such, CD-1 mice were used to characterize the murine pharmacokinetics of RVT-801, since Farber mice are frail, difficult to mate, and not numerous enough to conduct a full PK assessment.
  • systemic rhAC e.g., RVT-801
  • CD-1 mice approximates that in Farber mice, and may represent the minimum systemic levels of rhAC in Farber mice following a single dose of RVT-801 administered intraperitoneally.
  • Recombinant human AC e.g., RVT-801
  • a Farber disease murine model e.g., liver, spleen, and lung.
  • Measurement of systemic levels of recombinant human AC could, therefore, potentially underestimate exposure in relevant tissue of human subjects suffering from Farber disease.
  • relying on simple allometry to scale a dose in nonclinical species to humans may not provide adequate estimates of tissue exposure to impart efficacy.
  • HED human equivalent dose
  • FDA United States Food & Drug Administration
  • BSA body surface area
  • CDER Center for Drug Evaluation and Research
  • HED estimate approaches described herein also consider physiological factors including, but not limited to, major clearance mechanisms from the vasculature to tissues.
  • mice Data reported in this specification in mice indicate that the liver and spleen are major target tissues for the uptake of RVT-801 following systemic administration.
  • a HED may be derived by combining BSA scaling and the ratios of tissue mass to bodyweight (“BW”) in mice and humans. In this manner, it is possible to derive a dose scaling factor that accounts for the relative size of each compartment of recombinant human AC (RVT-801) distribution in each species.
  • RVT-801 recombinant human AC
  • a method of treating a human adult subject with Farber disease comprising administering to the human subject recombinant human acid ceramidase (rhAC) at a dose of about 0.8 mg/kg, is provided.
  • rhAC human acid ceramidase
  • Also provided in an embodiment is a method of treating a human child with Farber disease, the method comprising administering to the child recombinant human acid ceramidase (rhAC) at a dose of about 1.2 mg/kg.
  • rhAC human acid ceramidase
  • Also provided in an embodiment is a method of treating a human child with Farber disease, the method comprising administering to the child recombinant human acid ceramidase (rhAC) at a dose of about 2 mg/kg.
  • rhAC human acid ceramidase
  • Also provided in an embodiment is a method of treating a human child with Farber disease, the method comprising administering to the child recombinant human acid ceramidase (rhAC) at a dose of about 5 mg/kg.
  • rhAC human acid ceramidase
  • a method of treating a human subject with Farber disease may comprise administering to the human subject a therapeutically effective dose of recombinant human acid ceramidase (rhAC) that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse one or more of: T max of about 0.25 to 1.0 hours, C max of about 1.23 to about 2.17 ⁇ g/mL, or area under the curve (AUC) of about 1.37 hr* ⁇ g/mL to about 1.49 hr* ⁇ g/mL (or about 1 hr* ⁇ g/mL to about 2 hr* ⁇ g/mL), as disclosed below in this specification.
  • MED maximum effective dose
  • the maximum effective dose (MED) in Farber mice was determined to be 10 mg/kg delivered by bolus IP injection.
  • the value for AUCiast determined following a single 10 mg/kg dose to aged-matched, wild-type CD-1 mice may be 1.37 ⁇ g.h/mL with a C max of 1.23 ⁇ g/mL.
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse ratios greater than 1 for AUC liver :AUC serum , AUC spleen :AUC serum , AUC lung :AUC serum , AUC kidney :AUC serum , AUC heart :AUC serum .
  • MED maximum effective dose
  • rhAC rhAC
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse one or more of: an AUC liver :AUC serum ratio of about 92.9, an AUC spleen :AUC serum ratio of about 47.6, an AUC lung :AUC serum ratio of about 5.64, an AUC kidney :AUC serum ratio of about 17.3, an AUC heart :AUC serum ratio of about 2.69, an AUC whole blood :AUC serum ratio of about 0.575, an AUC brain :AUC serum ratio of about 0.0281, or an AUC BALF :AUC serum ratio of about 0.000165 in mice (where BALF is broncho
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse ratios greater than 1 for AUC liver :AUC serum , AUC spleen :AUC serum , AUC lung :AUC serum , AUC kidney :AUC serum , AUC heart :AUC serum , AUC whole blood :AUC serum , AUC brain :AUC serum .
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a T max of about 0.25 to 1.0 hours.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max of about 1.23 ⁇ g/mL to 2.17 ⁇ g/mL.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse an AUC of about 1.37 to 1.49 hr* ⁇ g/mL.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max , or AUC 0-last or AUC tissue /AUC serum in tissue or matrix in accordance with the values set forth in FIG. 7 .
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse an AUCo to last of about 138 hr* ⁇ g/mL in liver tissue.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max for the drug of about 13 ⁇ g/mL in liver tissue.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse an AUCo to last of about 70.9 hr* ⁇ g/mL in splenic tissue
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max for the drug of about 7.58 ⁇ g/mL in splenic.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse an AUC 0 to last of about 25.8 hr* ⁇ g/mL in kidney tissue.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max for the drug of about 2.61 ⁇ g/mL in kidney tissue.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse an AUCo to last of about 4.01 hr* ⁇ g/mL in heart tissue.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max for the drug of about 0.362 ⁇ g/mL in heart tissue.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse an AUC 0 to last of about 0.0419 hr* ⁇ g/mL in brain tissue.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max of about 0.147 ⁇ g/mL in brain.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse AUC 0 to last of about 0.858 hr* ⁇ g/mL in blood.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max for the drug of about 1.23 ⁇ g/mL in blood.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the human subject a therapeutically effective dose of recombinant human acid ceramidase (rhAC) that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, that provides AUCo to last of about 0.000245 hr* ⁇ g/mL in BALF.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max for the drug of about 0.00196 ⁇ g/mL in BALF.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse an AUCo to last of about 8.4 hr* ⁇ g/mL in lung tissue.
  • MED maximum effective dose
  • a method of treating a human subject with Farber disease may comprise administering to the subject a therapeutically effective dose of rhAC that may be determined based on the pharmacokinetic profile, obtained in juvenile, healthy CD-1 mice receiving a single, maximum effective dose (“MED”) of 10 mg/kg dose of rhAC (i.e., RVT-801) intraperitoneally, wherein the rhAC elicits in the healthy CD-1 mouse a C max for the drug of about 1.42 ⁇ g/mL in lung tissue.
  • MED maximum effective dose
  • FIG. 1 shows a time course of serum RVT-801 levels in healthy, juvenile CD-1 mice, age-matched to Farber disease mice treated with RVT-801, after a single injection of RVT-801 at doses of 1, 3, and 10 mg/kg according to Example 1.
  • FIG. 2 lists pharmacokinetic metrics of serum RVT-801 in healthy, juvenile CD-1 mice after a single injection of RVT-801 at doses of 1, 3, and 10 mg/kg according to Example 1.
  • FIG. 3 shows RVT-801 concentrations in healthy age-matched CD-1 mouse serum and rhAC activity in plasma of Farber disease mice after a single injection of RVT-801 at a dose of 10 mg/kg according to Example 1, showing comparability between Farber mouse and age-matched parental CD-1 strain mouse pharmacokinetic profiles.
  • FIG. 4 shows a time course of RVT-801 concentrations in serum and various tissues (liver, spleen, kidney, heart, lung, and brain) of healthy, juvenile CD-1 mice after a single injection of serum RVT-801 at a dose of 10 mg/kg according to Example 2.
  • FIG. 5A shows a time course of RVT-801 concentrations in liver tissue of healthy, juvenile CD-1 mice after a single injection of RVT-801 at doses of 1, 3, and 10 mg/kg according to Example 2.
  • FIG. 5B shows a time course of RVT-801 concentrations in spleen tissue of healthy, juvenile CD-1 mice after a single injection of serum RVT-801 at doses of 1, 3, and10 mg/kg according to Example 2.
  • FIG. 6A compares AUC 0-last in serum, whole blood, liver tissue extract, and spleen tissues of healthy, juvenile CD-1 mice after a single injection of serum RVT-801 at doses of 1, 3, and10 mg/kg according to Example 2.
  • FIG. 6B shows AUC tissue /AUC serum AUC ratios calculated from RVT-801 concentrations in serum, along with hepatic and spleenic tissues of healthy, juvenile CD-1 mice after a single injection of RVT-801 at doses of 1, 3, and 10 mg/kg according to Example 2.
  • FIG. 7 shows C max, AUC 0-last , and AUC tissue /AUC serum calculated from RVT-801 concentrations in serum, whole blood, and various tissue extracts (liver, spleen, kidney, lung, heart, brain and bronchoalveolar lavage fluid (BALF) of healthy, juvenile CD-1 mice after a single injection of RVT-801 at a dose of 10 mg/kg according to Example 2.
  • tissue extracts liver, spleen, kidney, lung, heart, brain and bronchoalveolar lavage fluid (BALF) of healthy, juvenile CD-1 mice after a single injection of RVT-801 at a dose of 10 mg/kg according to Example 2.
  • FIG. 8 shows tissue distribution and comparison of AUC tissue /AUC serum in various tissues of healthy, juvenile CD-1 mice after a single injection of RVT-801 at a dose of 10 mg/kg according to Example 2.
  • FIG. 9 shows estimated human equivalent doses (HEDs) scaled by body surface area (BSA), estimated from the MED with respect to reduction of accumulated ceramides in liver and spleen of 10 mg/kg in Farber mice according to Example 3.
  • HEDs human equivalent doses scaled by body surface area
  • FIG. 10 shows tissue-specific HEDs, scaled by tissue-to-body weight ratio, estimated from the MED of 10 mg/kg in Farber mice with respect to reduction of accumulated ceramides in liver and spleen according to Example 3.
  • FIG. 11 shows results of both (1) BSA-based HED calculations and (2) tissue-to-body weight ratio-based HED calculations according to Example 3.
  • FIG. 12 shows comparison of an embodiment of HED for RVT-801 in the present application to the doses of approved enzyme replacement therapies (ERTs).
  • ERTs enzyme replacement therapies
  • FIG. 13 shows mean rhAC concentration-time data in liver, spleen, and kidney of Farber mice following a single or repeat weekly 10 mg/kg dose of RVT-801.
  • FIG. 14 shows data on rhAC serum concentrations in healthy age-matched CD-1 mice and acid ceramidase plasma activity in Farber mice following a single 10 mg/kg dose of RVT-801.
  • FIG. 15 shows final rhAC tissue:serum AUC ratio in healthy juvenile CD-1 mice following a single 10 mg/kg dose of RVT-801.
  • FIG. 16 shows rhAC in circulation following single 10 mg/kg IP bolus injection of RVT-801 to CD-1 and Farber mice.
  • FIG. 17 shows rhAC concentrations in circulation and in key tissues following a single 10 mg/kg bolus IP injection of RVT-801 to CD-1 and Farber mice.
  • FIGS. 19A-G present the mean rhAC concentrations in circulation ( FIG. 19A ), liver ( FIG. 19B ), spleen ( FIG. 19C ), kidney ( FIG. 19D ), heart ( FIG. 19E ), lung ( FIG. 19F ), and brain ( FIG. 19G ) tissue in Farber mice (open symbols) and age-matched CD-1 mice (filled symbols) following either a single 10 mg/kg dose or multiple once-weekly doses at a 10 mg/kg/dose of RVT-801 administered via bolus IP injection, in accordance with Example 5.
  • FIGS. 20A and 20B show mean rhAC tissue concentration-time profiles following IP administration to juvenile CD-1 mice in RVT-801-9013 Part B (Linear FIG. 20A and Log-Linear FIG. 20B ), respectively.
  • FIG. 21 presents RVT-801 tissue:serum exposure ratios in BALF, blood, brain, heart, kidney, liver, lung, and spleen based on AUC last following single doses of RVT-801 of 1mg/kg, 3 mg/kg, and 10 mg/kg to juvenile CD-1 mice.
  • FIGS. 22A-D presents dose normalized AUC plotted verses dose level for various tissues following IP administration of RVT-801 at doses of 1 mg/kg, 3 mg/kg and 10 mg/kg in juvenile CD-1 mice.
  • the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ⁇ 10% and remain within the scope of the disclosed embodiments.
  • ASM activity refers to a related lipid hydrolase that tightly binds to AC and co-purifies with it (Bernardo, K., R. Hurwitz, T. Zenk, R. J. Desnick, K. Ferlinz, E. H. Schuchman, and K. Sandhoff, 1995, “Purification, characterization, and biosynthesis of human acid ceramidase,” J Biol Chem, 270:11098-11102).
  • animal includes, but is not limited to, humans and non-human vertebrates such as wild, domestic, and farm animals.
  • the animal can also be referred to as a “subject.”
  • carrier means a diluent, adjuvant, or excipient with which a compound is administered.
  • Pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents can be used.
  • child refers to an age that is from newborn to 18 years old.
  • the terms “comprising” (and any form of comprising, such as “comprise”, “comprises”, and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Additionally, a term that is used in conjunction with the term “comprising” is also understood to be able to be used in conjunction with the term “consisting of” or “consisting essentially of.”
  • contacting means bringing together of two elements in an in vitro system or an in vivo system.
  • “contacting” rhAC polypeptide an individual, subject, or cell includes the administration of the polypeptide to an individual or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing the polypeptide.
  • contacting can refer to transfecting or infecting a cell with a nucleic acid molecule encoding the polypeptide.
  • “Farber mouse,” “Farber mice,” “Farber disease mice,” and “Farber disease mouse” means severe Farber disease mouse model (Asah1 P361R/P31R6 ).
  • the Farber mouse is a murine model based on a severe Farber disease patient genotype. Based on the severe Farber disease patient genotype, knock-in mice that are homozygous for a single nucleotide Asah1 P361R/P361R mutation were derived from a mixed genetic background colony (W4/129Sv/CD-1) (“CD-1 mice”) to establish a murine model of severe Farber disease, as previously described (Alayoubi, A. M., J. C. Wang, B. C. Au, S.
  • Farber mice produce an altered AC, incapable of hydrolyzing ceramides to their sphingosine and fatty acids constituents. These Farber mice exhibit features characteristic of clinical Farber disease including disruption of bone formation and the morphology of intra-articular tissues, presence of lipid-laden macrophages, and systemic inflammation, along with a significantly stunted rate of growth and shortened lifespan compared to their wild-type (Asah1 WT/WT ) or heterozygous (Asah1 WT/P361R ) littermates.
  • an “effective amount” of an enzyme delivered to a subject is an amount sufficient to improve the clinical course of a Farber disease where clinical improvement is measured by any of the variety of defined parameters well known to the skilled artisan.
  • the terms “subject,” “individual” or “patient,” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.
  • the phrase “in need thereof” means that the subject has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the subject can be in need thereof.
  • integer from X to Y means any integer that includes the endpoints.
  • integer from 1 to 5 means 1, 2, 3, 4, or 5.
  • isolated means that the compounds described herein are separated from other components of either (a) a natural source, such as a plant or cell, or (b) a synthetic organic chemical reaction mixture, such as by conventional techniques.
  • the term “mammal” means a rodent (i.e., a mouse, a rat, or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or a human. In some embodiments, the mammal is a human.
  • pharmaceutically acceptable means those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with tissues of humans and animals.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • serum means any blood-derived matrix, including, without limitation, “serum,” “plasma,” or “whole blood.”
  • substantially isolated means a compound that is at least partially or substantially separated from the environment in which it is formed or detected.
  • the phrase “therapeutically effective amount” means the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor, or other clinician.
  • the therapeutic effect is dependent upon the disorder being treated or the biological effect desired.
  • the therapeutic effect can be a decrease in the severity of symptoms associated with the disorder and/or inhibition (partial or complete) of progression of the disorder, or improved treatment, healing, prevention or elimination of a disorder, or side-effects.
  • the amount needed to elicit the therapeutic response can be determined based on the age, health, size and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject's response to treatment.
  • Clinical monitors of disease status may include but are not limited to ceramide levels, weight, joint length, inflammation, or any other clinical phenotype known to be associated with Farber disease.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease.
  • treatment of Farber disease” or “treating Farber disease” means an activity that alleviates or ameliorates any of the primary phenomena or secondary symptoms associated with Farber disease or other condition described herein.
  • methods of treating Farber disease are provided.
  • the subject is a subject in need thereof.
  • the subject in need thereof is diagnosed with Farber disease.
  • the subject is also identified as having: 1) subcutaneous nodules; 2) an acid ceramidase activity value in white blood cells, cultured skin fibroblasts, or other biological sources (e.g., plasma) that is less than 30% of control values; and/or 3) nucleotide changes within both alleles of the acid ceramidase gene (ASAH1) that indicate, through bioinformatic, gene expression studies, and/or other methods, a possible loss of function of the acid ceramidase protein.
  • SSH1 acid ceramidase activity value in white blood cells, cultured skin fibroblasts, or other biological sources
  • the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.1 mg/kg to about 50 mg/kg. In some embodiments, the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg. In some embodiments, the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg.
  • the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • methods of reducing lipogranulomas in a subject with, or suspected of having, Farber disease are provided.
  • the subject is a subject in need thereof.
  • the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.1 mg/kg to about 50 mg/kg.
  • the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • methods of reducing absolute or normalized spleen weight in a subject with, or suspected of having, Farber disease are provided.
  • the subject is a subject in need thereof.
  • the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.1 mg/kg to about 50 mg/kg.
  • the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • methods of reducing ceramide levels in a subject with, or suspected of having, Farber disease are provided.
  • the subject is a subject in need thereof.
  • the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.1 mg/kg to about 50 mg/kg.
  • the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • Reducing ceramide can also refer to decreasing ceramide or increasing the metabolizing of ceramide, which would lead to reduced ceramide levels.
  • methods of increasing sphingosine levels in a subject with, or suspected of having, Farber disease are provided.
  • the subject is a subject in need thereof.
  • the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.1 mg/kg to about 50 mg/kg.
  • the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 ,g/kg or 10 mg/kg.
  • the pharmaceutical composition is a solution.
  • the pharmaceutical composition comprises cell conditioned media comprising the rhAC.
  • the term “cell conditioned media” refers to cell culture media that has been used to culture cells expressing rhAC and where the protein is secreted into the media and then the protein is isolated or purified from the media.
  • the media is used to treat the subject.
  • the media for example, can be applied to the skin of a subject to treat any of the conditions, symptoms, or disorders described herein.
  • the pharmaceutical composition is administered by contacting the skin of the subject.
  • the administration is parenteral administration.
  • the administration comprises injecting the pharmaceutical composition to the subject.
  • the administration is an intraperitoneal injection or intravenous injection.
  • the administration is oral administration.
  • methods of treating Farber disease in a subject in need thereof comprising expressing recombinant human acid ceramidase (rhAC) in a cell; isolating the expressed rhAC from the cell; and administering to the subject a pharmaceutical composition comprising the isolated expressed rhAC in an effective amount of about 0.1 mg/kg to about 50 mg/kg.
  • the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • methods of reducing lipogranulomas in a subject with, or suspected of having, Farber disease comprising expressing recombinant human acid ceramidase (rhAC) in a cell; isolating the expressed rhAC from the cell; and administering to the subject a pharmaceutical composition comprising the isolated expressed rhAC in an effective amount of about 0.1 mg/kg to about 50 mg/kg.
  • the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments, the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • the method comprises treating lipogranulomatosis (now, Farber disease) in a subject with, or suspected of having, Farber disease are provided, the methods comprising expressing recombinant human acid ceramidase (rhAC) in a cell; isolating the expressed rhAC from the cell; and administering to the subject a pharmaceutical composition comprising the isolated expressed rhAC in an effective amount of about 0.1 mg/kg to about 50 mg/kg. In some embodiments, the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • rhAC recombinant human acid ceramidase
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • methods of reducing spleen weight (absolute or normalized to body weight) in a subject with, or suspected of having, Farber disease comprising expressing recombinant human acid ceramidase (rhAC) in a cell; isolating the expressed rhAC from the cell; and administering to the subject a pharmaceutical composition comprising the isolated expressed rhAC in an effective amount of about 0.1 mg/kg to about 50 mg/kg.
  • the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • methods of reducing ceramide in a subject with, or suspected of having, Farber disease comprising expressing recombinant human acid ceramidase (rhAC) in a cell; isolating the expressed rhAC from the cell; and administering to the subject a pharmaceutical composition comprising the isolated expressed rhAC in an effective amount of about 0.1 mg/kg to about 50 mg/kg.
  • the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • methods of increasing sphingosine in a subject with, or suspected of having, Farber disease comprising expressing recombinant human acid ceramidase (rhAC) in a cell; isolating the expressed rhAC from the cell; and administering to the subject a pharmaceutical composition comprising the isolated expressed rhAC in an effective amount of about 0.1 mg/kg to about 50 mg/kg.
  • the methods comprising administering to a human adult subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 0.8 mg/kg.
  • the methods comprising administering to a human child a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1.2 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 1 mg/kg to about 5 mg/kg. In some embodiments the methods comprising administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount of about 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.
  • the expressing recombinant human acid ceramidase (rhAC) in a cell comprises transferring a vector encoding rhAC into the cell.
  • the vector comprises a nucleic acid molecule encoding rhAC.
  • the nucleic acid molecule is a molecule as described herein or any other nucleic acid molecule that encodes the rhAC polypeptide or homolog thereof, which is described in more detail herein.
  • the vector is a viral vector.
  • the vector can be a retroviral vector or a DNA virus vector, such as adenovirus, AAV, and the like.
  • the vector is a plasmid.
  • the vector comprises a promoter operably linked to the rhAC.
  • the promoter is a constitutive promoter.
  • the promoter is the SV40 promoter, CMV promoter, EF1 alpha promoter, or any combination thereof, or any other promoter that is active in a mammalian cell.
  • the vector is transfected or infected into the cell.
  • the methods of introducing the vector in the cell are not critical and any method can be used to provide sufficient expression of the rhAC polypeptide in the cell.
  • the cell is a mammalian cell. In some embodiments, the cell is not a human cell. In some embodiments, the cell is a hamster cell. In some embodiments, the cell is a Chinese hamster ovarian (CHO) cell. In some embodiments, the cell can be grown in a serum-free or substantially free of serum environment. In some embodiments, the cell is derived from a CHO-K1 cell. In some embodiments, the cell is a murine cell. In some embodiments, the cell is a murine myeloma cell. In some embodiments, the cell is a NSO cell. In some embodiments, the effective amount that is administered is as described herein, above and below.
  • the pharmaceutical composition is administered as described herein.
  • the composition is administered to a subject orally, by inhalation, by intranasal instillation, topically, transdermally, parenterally, subcutaneously, intravenous injection, intra-arterial injection, intramuscular injection, intraplurally, intraperitoneally, intrathecally, or by application to a mucous membrane.
  • rhAC refers to recombinant human acid ceramidase encoded by the ASAH1 gene (NCBI UniGene GeneID No. 427). AC hydrolyzes the amide bond linking the sphingosine and fatty acid moieties of the lipid ceramide (Park, J.-H., Schuchman E. H., 2006, “Acid ceramidase and human disease,” Biochim. Biophys. Acta. 1758(12): 2133-2138; Nikolova-Karakashian et al., 2000, “Ceramidases,” Methods Enzymol.
  • ceramidases There are three types of ceramidases described to date (Nikolova-Karakashian, 2000). These are classified as acid, neutral, and alkaline ceramidases according to their pH optimum of enzymatic activity. ACs have optimal enzymatic activity at a pH ⁇ 5.
  • the human AC was the first ceramidase to be cloned (Koch et al., 1996, “Molecular Cloning and Characterization of a Full-length Complementary DNA Encoding Human Acid Ceramidase: Identification Of The First Molecular Lesion Causing Farber Disease,” J. Biol. Chem. 271:33110-33115). It is localized in the lysosome and is mainly responsible for the catabolism of ceramide.
  • AC (N-acylsphingosine deacylase, I.U.B.M.B. Enzyme No. EC 3.5.1.23) protein has been purified from several sources, and the human and mouse cDNAs and genes have been obtained. See Bernardo et al., J. Biol. Chem. 270:11098-102 (1995); Koch et al., J. Biol. Chem. 2711:33110-5 (1996); Li et al., Genomics 50:267-74 (1998); Li et al., Genomics 62:223-31 (1999). It is produced through cleavage of the AC precursor protein (see Ferlinz et al., J. Biol. Chem.
  • the AC alpha subunit begins at the amino acid at position 22 and continues through position 142 (as shown in bold in SEQ ID NO: 1 in the Table of Sequences), while the beta subunit of the AC begins with the amino acid at position 143 and continues through position 395 (as shown in italics in SEQ ID NO: 1).
  • active acid ceramidase refers to AC precursor proteins that have undergone autoproteolytic cleavage into the active form (composed of ⁇ - and ⁇ -subunits).
  • the mechanism of human AC cleavage and activation is reported in (Shtraizent, N., E. Eliyahu, J. H. Park, X. He, R. Shalgi and E. H. Schuchman, 2008, “Autoproteolytic cleavage and activation of human acid ceramidase,” J Biol Chem, 283(17):11253-11259).
  • Activation is also promoted by the intracellular environment, and, based on highly conserved sequences at the cleavage site of ceramidase precursor proteins across species, is expected to occur in most, if not all, cell types.
  • inactive acid ceramidase As used herein, “inactive acid ceramidase,” “inactive AC,” or “inactive acid ceramidase precursor,” “inactive AC precursor,” or (AC preprotein) refers to AC precursor protein that has not undergone autoproteolytic cleavage into the active form.
  • Inactive AC precursors and active ACs suitable for use in the recombinant acid ceramidase of this and all aspects of the present invention can be homologous (i.e., derived from the same species) or heterologous (i.e., derived from a different species) to the tissue, cells, and/or subject being treated.
  • Acid ceramidase (e.g., AC) precursor proteins undergo autoproteolytic cleavage into the active form (composed of ⁇ - and ⁇ -subunits).
  • AC Acid ceramidase
  • the mechanism of human AC cleavage and activation is reported in (Shtraizent, 2008). This is promoted by the intracellular environment, and, based on highly conserved sequences at the cleavage site of ceramidase precursor proteins across species, is expected to occur in most, if not all, cell types.
  • ceramidase as used herein includes both active ceramidases and ceramidase precursor proteins, where ceramidase precursor proteins are converted into active ceramidase proteins through autoproteolytic cleavage.
  • Embodiments in which the precursor protein is taken up by the cell of interest and converted into active ceramidase thereby, as well as embodiments in which the precursor protein is converted into active ceramidase by a different cell or agent (present, for example, in a culture medium), are both contemplated.
  • Active ACs and inactive AC precursor proteins that can be used in this and all aspects of the present invention include, without limitation, those set forth in Table 1 of US 2016/0038574, the contents of which are hereby incorporated by reference.in their entirety.
  • the rhAC comprises an amino acid sequence of SEQ ID NO: 1.
  • RVT-801 is a recombinant human acid ceramidase (rhAC) in activated form for the treatment of Farber disease. The alpha and beta subunits of the activated rhAC are joined by a disulfide bond.
  • the molecule is a recombinant human acid ceramidase (rhAC) derived from CHO-M cells transfected with a DNA plasmid vector expressing rhAC.
  • RVT-801 is based on UniProtKB Code: Q13510.
  • RVT-801rhAC has been established in a murine model of severe Farber disease (He, et al, 2017) and has been characterized over multiple studies with endpoints describing positive impacts on histopathological and immunological outcomes along with concomitant reduction of accumulated ceramides.
  • the rhAC is a protein that is a homolog of SEQ ID NO: 1. In some embodiments, the rhAC is encoded by a nucleic acid molecule of SEQ ID NO: 2. In some embodiments, the rhAC is encoded by a nucleic acid molecule of SEQ ID NO: 3. In some embodiments, the rhAC is encoded by a nucleic acid molecule of SEQ ID NO: 4. In some embodiments, the sequence is as defined in GenBank accession number NM_177924.3 or NM_177924.4, each of which is incorporated by reference in its entirety.
  • the nucleotide sequence encoding the protein can be the complete sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or be simply the coding region of the sequence
  • the coding region could be nucleotides 313 to 1500 of SEQ ID NO: 2 or the corresponding coding region found in SEQ ID NO: 3 or SEQ ID NO: 4.
  • the genetic code is degenerate and, therefore other codons can be used to encode the same protein without being outside of what is disclosed. Since the amino acid sequence is known any nucleotide sequence that encodes the amino acid sequence is acceptable.
  • the nucleotide sequence comprises a signal peptide.
  • the signal peptide is an amino acid sequence encoded by nucleotides 313 to 375 of SEQ ID NO: 2.
  • the protein that is produced comprises a signal peptide of amino acid residues 1-21 of SEQ ID NO: 1.
  • the protein that is produced does not comprises a signal peptide, such as the signal peptide of amino acid residues 1-21 of SEQ ID NO: 1.
  • the signal peptide is removed during a post-translational processing where the enzyme is processed into its different subunits.
  • the nucleotide sequence is codon optimized for the cell that it the protein is being expressed from.
  • the protein comprises an alpha-subunit, a beta-subunit, and the like.
  • the protein that is produced comprises a peptide of amino acid residues 22-142, 45-139, 134-379, 143-395, or 1-395 of SEQ ID NO: 1.
  • the peptide can be a single protein or a polypeptide of different sequences to form the enzyme.
  • the protein is free of amino acid residues 1-21. These regions can be encoded by a single nucleotide sequence or separate nucleotide sequences or a combination of nucleotide sequences. As discussed herein, any nucleotide sequence encoding the protein can be used and is not limited to the sequence described herein as SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • the rhAC has acid ceramidase (AC) activity but does not have any detectable acid sphingomyelinase activity, such as the rhAC produced in Examples 1 and 2 below.
  • the acid sphingomyelinase activity may be removed, for example, by heat inactivation. Heat inactivation may also remove other contaminating proteins from an rhAC preparation. See, e.g., U.S. Patent Application Publication No. 20160038574, which is incorporated herein in its entirety.
  • homolog refers to protein sequences having between 80% and 100% sequence identity to a reference sequence. Percent identity between two peptide chains can be determined by pair wise alignment using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen Corp., Carslbad, Calif). In some embodiments, the homolog has at least, or about, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to a sequence described herein, such as SEQ ID NO: 1.
  • the protein delivered to the subject conservative substitutions as compared to a sequence described herein.
  • Non-limiting exemplary conservative substitutions are shown in Table 3 are encompassed within the scope of the disclosed subject matter. Substitutions may also be made to improve function of the enzyme, for example stability or enzyme activity. Conservative substitutions will produce molecules having functional and chemical characteristics similar to those molecules into which such modifications are made. Exemplary amino acid substitutions are shown in Table 3 below.
  • the protein is produced from a cell.
  • the cell is a Chinese Hamster Ovarian cell, “CHO cell.”
  • a nucleic acid sequence encoding the proteins described herein can be genomic DNA or cDNA, or RNA (e.g. mRNA) which encodes at least one of proteins described herein.
  • RNA e.g. mRNA
  • the use of cDNA requires that gene expression elements appropriate for the host cell be combined with the gene in order to achieve synthesis of the desired protein.
  • the use of cDNA sequences can advantageous over genomic sequences (which contain introns), in that cDNA sequences can be expressed in bacteria or other hosts which lack appropriate RNA splicing systems.
  • One of skill in the art can determine the best system for expressing the protein.
  • the protein is produced according to U.S. Patent Application Publication No. 20160038574, which is incorporated by reference in its entirety.
  • the genetic code is degenerate, more than one codon can be used to encode a particular amino acid. Using the genetic code, one or more different oligonucleotides can be identified, each of which would be capable of encoding the amino acid sequences described herein.
  • the enzyme that is administered to the subject to treat Farber disease or a condition associate therewith can be purified.
  • purified with referenced to a protein refers to a protein that is substantially free of other material that associates with the molecule in its natural environment.
  • a purified protein is substantially free of the cellular material or other proteins from the cell or tissue from which it is derived.
  • the term refers to preparations where the isolated protein is sufficiently pure to be analyzed, or at least 70% to 80% (w/w) pure, at least 80%-90% (w/w) pure, 90-95% pure; and, at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.
  • the protein is purified from a cell, such as but not limited to a CHO cell.
  • embodiments provided herein provide methods of treating Farber disease.
  • the methods comprise administering a therapeutically or prophylactically effective amount of one or more proteins described herein to a subject with Farber disease or suspected of having Farber disease.
  • Treatment of subjects may comprise the administration of a therapeutically effective amount of the proteins described herein.
  • the proteins can be provided in a kit as described herein.
  • the proteins can be used or administered alone or in admixture with an additional therapeutic.
  • additional therapeutics include, but are not limited to, inhibitors of acid sphingomyelinase (e.g., amitriptyline (Becker et al., 2010, “Acid Sphingomyelinase Inhibitors Normalize Pulmonary Ceramide and Inflammation in Cystic Fibrosis,” Am. J. Respir. Cell. Mol.
  • ceramide synthases e.g., Shiffmann, S., Hartmann, D., Birod, K., Ferreir ⁇ s, N., Schreiber, Y., Zivkovic, A., Geisslinger, G., Grösch, S., and Stark, H., 2012, “Inhibitors of Specific Ceramide Synthases,” Biochimie, 94(2):558-565, which is hereby incorporated by reference in its entirety)).
  • the additional therapeutic can also be ceramidase mixtures described in U.S. Patent Application Publication No. 20160038574, which is hereby incorporated by reference in its entirety.
  • methods of treating Farber disease in a subject in need thereof comprise administering to the subject a pharmaceutical composition comprising a recombinant human acid ceramidase in an effective amount about once a week, once every 2, 3, or 4 weeks, or once a month, for about 10, about 20, or about 30 weeks, 1, 5, 10, or 25 years, or the duration of a patient's life.
  • Suitable vehicles and their formulation and packaging are described, for example, in Remington: The Science and Practice of Pharmacy (21st ed., Troy, D. ed., Lippincott Williams & Wilkins, Baltimore, Md. (2005) Chapters 40 and 41). Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved through the use of polymers to complex or absorb the compounds. Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
  • microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate)-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • a dosage of protein which is in the range of from about 1 ng/kg-100 ng/kg, 100 ng/kg-500 ng/kg, 500 ng/kg-1 ⁇ g/kg, 1 ⁇ g /kg-100 ⁇ g /kg, 100 ⁇ g/kg-500 ⁇ g /kg, 500 ⁇ g /kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100 mg/kg-500 mg/kg (body weight of recipient), although a lower or higher dosage may be administered.
  • the human dose of rhAC in adults is about 0.8 mg/kg. In some embodiments, the human dose of rhAC in children is about ⁇ 1.2 mg/kg.
  • the effective amount of rhAC that is administered is about 0.1 mg/kg to about 10 mg/kg. In some embodiments, the effective amount is about 1 mg/kg to about 5 mg/kg. In some embodiments, the effective amount is about 10 mg/kg to about 50 mg/kg. In some embodiments, the effective amount is about 10 mg/kg to about 20 mg/kg. In some embodiments, the effective amount is about 20 mg/kg to about 30 mg/kg. In some embodiments, the effective amount is about 30 mg/kg to about 40 mg/kg. In some embodiments, the effective amount is about 40 mg/kg to about 50 mg/kg. In some embodiments, the effective amount is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg.
  • the dosage can be administered once a day, twice a day, three times a day, four times a day, once a week, twice a week, once every two weeks, or once a month.
  • the dose is administered once a week.
  • the dose is administered once every two weeks.
  • the treatment may also be given in a single dose schedule, or a multiple dose schedule in which a primary course of treatment may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the response, for example, once a week for 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months.
  • Suitable treatment schedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired responses expected to reduce disease symptoms, or reduce severity of disease.
  • Other treatment schedules such as, but not limited to, those described above, can also be used.
  • the treatment is started when the subject is newborn, under 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age, or between 1 and 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 60, 70, or 80 years of age (e.g., between 1 and 2, between 1 and 3, etc.).
  • the subject is between 16 and 61. In some embodiments, the subject starts treatment at age 16. In some embodiments, the subject is between 12 and 69. In some embodiments, the subject starts treatment at age 12. In some embodiments, the subject is between 19 and 74. In some embodiments, the subject starts treatment at age 19. In some embodiments, the subject is between 4 and 62. In some embodiments, the subject starts treatment at age 4. In some embodiments, the subject is between 7 and 42.
  • the subject starts treatment at age 7. In some embodiments, the subject is newborn. In some embodiments, the subject is between 1 and 6 months. In some embodiments, the subject starts treatment at age 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. In some embodiments, the subject is between 6 and 43. In some embodiments, the subject starts treatment at age 6. In some embodiments, the subject is between 5 and 31. In some embodiments, the subject starts treatment at age 5. In some embodiments, the subject is between 5 and 57. In some embodiments, the subject is between 5 and 29. In some embodiments, the subject is between 1 and 3. In some embodiments, the subject starts treatment at age 1. In some embodiments, the subject is between 10 and 70. In some embodiments, the subject starts treatment at age 10. In some embodiments, the subject is between 5 and 80, between 10 and 70, between 20 and 75, between 5 and 60, or between 5 and 30 years of age.
  • a subject diagnosed with Farber disease is administered rhAC at about 1 mg/kg to about 5 mg/kg rhAC or about 1 mg/kg to about 5 mg/kg rhAC every two weeks.
  • the dosage escalates from 1 mg/kg or 2 mg/kg to 5 mg/kg at week 4. If a dose level is not tolerated by an individual subject, the dose for that subject may be reduced from 2 mg/kg to 1 mg/kg, or 5 mg/kg to 2 mg/kg, as appropriate.
  • the rhAC may be administered every 2 weeks for at least 10, 20, or 30 weeks or for the duration of the subject's life.
  • a subject is diagnosed with Farber disease and is identified as having: 1) subcutaneous nodules; and/or 2) an acid ceramidase activity value in white blood cells, cultured skin fibroblasts or other biological sources (e.g., plasma) that is less than 30% of control values; and/or 3) nucleotide changes within both alleles of the acid ceramidase gene (ASAH1) that indicate, through bioinformatic, gene expression studies, and/or other methods, a possible loss of function of the acid ceramidase protein.
  • the subject is administered rhAC every two weeks for 28 weeks.
  • the delivery of rhAC is by intravenous infusion (e.g., saline infusion). In some embodiments, starting at about 1 mg/kg and escalating to about 5 mg/kg rhAC (e.g., to 5 mg/kg at week 4).
  • the intravenous infusion may be 3 mL/hr. over time (generally 3.5 to 4 hours).
  • the dose is infused at a rate 3% of the total volume in the first hour and the remainder of the dose may be infused over the following 2.5 hours (i.e., infusion in the second hour may be approximately 40% of the nominal volume per hour until the dose is administered).
  • No maximum time of infusion is established in order to ensure that individual tolerability may be addressed on a case-by-case basis.
  • the infusion is administered through an in-line, low-protein binding, 0.2-micron filter.
  • the dose is normalized to weight in kg which will be determined at each dosing interval, based on the weight collected at the previous study visit.
  • site specific administration may be to body compartment or cavity such as intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, vaginal, rectal, oral, buccal, sublingual, intranasal, or transdermal means.
  • intraarticular intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramy
  • the therapeutic compositions described herein can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) or any other administration particularly in the form of liquid solutions or suspensions.
  • the formulation can also be suitable for an injectable formulation.
  • the injectable formulation is sterile.
  • the injectable formulation is pyrogen free.
  • the formulation is free of other antibodies that bind to other antigens other than an antigen described herein.
  • a protein of rhAC capable of treating Farber disease or other condition associated with rhAC activity or use to treat a rhAC related pathology is intended to be provided to subjects in an amount sufficient to affect a reduction, resolution, or amelioration in the related symptom or pathology.
  • a pathology includes the symptoms of Farber disease as described herein in a subject.
  • An amount is said to be sufficient or a “therapeutically effective amount” to “affect” the reduction of symptoms if the dosage, route of administration, and dosing schedule of the agent are sufficient to influence such a response.
  • Responses to the protein can be measured by analysis of subject's affected tissues, organs, or cells as by imaging techniques or by ex vivo analysis of tissue samples.
  • an agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • an amount is a therapeutically effective amount if it is an amount that can be used to treat, ameliorate, or inhibit symptoms of Farber disease that a subject is subject to. Non-limiting examples of such amounts are provided herein, but are not intended to be limited to such amount if context dictates another amount.
  • efficacy of treatment is assessed by any of the following means:
  • pharmacokinetics of RVT-801 following administration to Farber mice or healthy mice at different doses is assessed based on noncompartmental methods.
  • Noncompartmental pharmacokinetics methods estimate the exposure to a drug by estimating the area under the curve of a concentration-time graph, among others, with the follow metrics know in the art:
  • tissue-specific efficacy of treatment is assessed by determining tissue-specific pharmacokinetics of RVT-801 based on the above described noncompartmental pharmacokinetics methods.
  • HED Human Equivalent Dose
  • BSA body surface area
  • organ:bodyweight ratios between species for liver and spleen as the major tissues for ceramide accumulation and in which uptake of RVT-801 predominated.
  • PK studies demonstrated the mechanism for clearance from the vasculature is associated with uptake and/or distribution into tissues, thus the tissue weight-to-bodyweight ratio may impact dose and the BSA model may not be sufficiently predictive on its own.
  • HED MED* (Tissue human /BW human )/(Tissue mouse /BW mouse ) a 10 mg/kg dose in mouse is equivalent to a ⁇ 3-5 mg/kg dose in an adult human or a ⁇ 4-5 mg/kg dose in a 15 kg child based on liver and spleen in human adults and children.
  • HED may be determined by combining the two scaling approaches above.
  • the proteins can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • kits which are described herein and below, are also provided which are useful for carrying out embodiments described herein.
  • the kits comprise a first container containing or packaged in association with the above-described polypeptides.
  • the kit may also comprise another container containing or packaged in association solutions necessary or convenient for carrying out the embodiments.
  • the containers can be made of glass, plastic, or foil and can be a vial, bottle, pouch, tube, bag, etc.
  • the kit may also contain written information, such as procedures for carrying out the embodiments or analytical information, such as the amount of reagent contained in the first container means.
  • the container may be in another container apparatus, e.g. a box or a bag, along with the written information.
  • kits for treating Farber disease comprises at least one container comprising a rhAC polypeptide or a nucleic acid molecule encoding the same.
  • the kit comprises a container comprising a cell that is configured to express rhAC.
  • the cell is a CHO cell.
  • the kit comprises conditioned media from a cell that expresses rhAC.
  • the conditioned media is from a CHO cell.
  • the present Examples provide the disposition of rhAC in key tissue compartments following administration across a range of efficacious doses to allow subsequent pharmacokinetics and tissue distribution modeling, and, based on the efficacious dose and tissue exposures, further provide prediction of a human equivalent dose (HED), scaled by body surface area or organ:body weight ratios.
  • HED human equivalent dose
  • the serum pharmacokinetic profile of rhAC (RVT-801) following administration of RVT-801 by intraperitoneal (i.p.) injection at a range of efficacious doses were assessed in healthy, juvenile, wild-type CD-1 mice (parental strain of Asah1 P361R/P361R mice, (See, He et al., 2017, which is incorporated herein by reference in its entirety).
  • Production and characterization of rhAC (RVT-801) were conducted according to PCT/2018/052463 (not yet published), filed on September 24, 2018, which is incorporated herein by reference in its entirety.
  • mice Male CD-1 mice, aged approximately 3.5 weeks (juvenile), were administered RVT-801 as a single bolus IP injection at either 1, 3, or 10 mg/kg.
  • Blood samples for pharmacokinetic analysis were collected from three (3) animals per time point (pre-dose, 0.5, 1, 2, 3, 4, 6 post-injection) in each dose group by cardiac puncture or other approved means to generate the maximum volume blood sample from each mouse.
  • Each blood sample was processed in its entirety to serum immediately for RVT-801 concentration analysis by ELISA.
  • Sandwich enzyme-linked immunosorbent assay have been qualified for the quantification of RVT-801 in mouse serum.
  • the method uses an affinity-purified rabbit anti-recombinant human AC (rhAC, RVT-801) polyclonal antibody for capture, and an affinity-purified horseradish peroxidase (HRP)-conjugated rabbit anti-rhAC polyclonal antibody for detection.
  • RVT-801 is used to establish the standard curve and control ranges.
  • the signal generated is proportional to the concentration of RVT-801 in the sample within the defined range of the assay.
  • the detailed 2-day assay procedure is as follows: On day 1, a 96-well clear polystyrene plate was coated with 100 ⁇ L of Capture Reagent (1 ⁇ g/mL rabbit anti-rhAC in PBS), and incubated overnight at 2-8° C. All other steps were performed on Day 2. All incubations on Day 2 took place at room temperature (RT), with shaking ( ⁇ 300-400 rpm). Briefly, the plate was washed 3 times with 300 ⁇ L of Wash Buffer (1 ⁇ PBS containing 0.05% Tween-20, PBST) and tapped on paper towels after the final wash.
  • RT room temperature
  • PBST wash Buffer
  • the plate was blocked with 300 ⁇ L of Assay Buffer (Casein/TBS containing 0.05% Tween 20) per well for at least 1 hour and washed as described above. After washing, 100 ⁇ L of calibration standards, controls, and samples, diluted to the MRD (50-fold in Assay Buffer), were added to each well, and the plate was incubated for approximately 2 hours and washed as described above. Next, 100 ⁇ L of Detection Reagent (0.2 ⁇ g/mL HRP-conjugated rabbit anti-rhAC in Assay Buffer) were added to each well, and the plate was incubated for approximately 1 hour and washed as described above.
  • Assay Buffer Casein/TBS containing 0.05% Tween 20
  • Noncompartmental pharmacokinetics analysis was performed by means of Certara WinNonlin Phoenix Version 7.0. Exposures ratios and graphs were generated with Microsoft Excel and GraphPad Prism.
  • RVT-801-9013A focused on characterizing serum PK. The following PK parameters were observed.
  • RVT-801-9013B focused on tissue distribution of RVT-801 and tissue:serum exposure ratios. The following PK parameters were observed.
  • duration of RVT-801 i.p. injection, 10 mg/kg exposure in serum of wild-type CD-1 mice correlated closely with systemic acid ceramidase (AC) activity in Farber disease mice (a solid line with black circles; reproduced from International Application No. PCT/US18/13509, the disclosure of which is incorporated by reference in its entirety).
  • AC systemic acid ceramidase
  • RVT-801 pharmacokinetics and tissue specific distribution of RVT-801 were assessed in a variety of organ tissues of wild-type CD-1 mice.
  • Tissue homogenates were prepared in CHAPS buffer with the addition of a protease inhibitor cocktail by processing the samples with a Qiagen TissueLyser II (3 cycles of 3 minutes each at a frequency of 30 Hz). The tissue homogenates were diluted to a standard 1 mg/mL total protein concentration prior to ELISA detection. Due to low protein content BALF samples were processed for detection on the basis of volume rather than protein concentration.
  • ELISA method qualified for the quantification of RVT-801 mouse serum has been adapted for quantification in mouse tissues (blood, liver, spleen, kidney, heart, lung, and brain).
  • the modified method included preparation of tissue lysates, quantification of total protein using a bicinchoninic acid (BCA) assay, and preparation of RVT-801 standard curve in lysis buffer.
  • the adapted method had a minimum required dilution (MRD) of 50-fold only for mouse serum quality control samples. Standards and study samples were not subjected to the MRD. The signal generated was proportional to the concentration of RVT-801 in the sample.
  • the lower and upper limits of quantification of the adapted assay were 0.400 ng/mL and 24.5 ng/mL, respectively. Measured RVT-801 concentrations were corrected for tissue homogenization and lysate dilution, as appropriate.
  • ELF epithelial lining fluid
  • RVT-801 Major mechanism for clearance of RVT-801 from circulation is uptake into key tissues implicated in ceramide accumulations. As shown in FIG. 4 , RVT-801 achieves extensive distribution across various organ tissues (liver, spleen, kidney, lung and heart). Exposure of RVT-801 in key tissues is markedly greater than in serum and persists for an extended period after RVT-801 becomes unmeasurable in systemic circulation. This finding may support less frequent dosing than indicated based on serum exposure. The prolonged duration of exposure in key organ tissues (liver, spleen) following acute RVT-801 treatment correlates to maximal reduction of tissue ceramides occurring beyond the duration of systemic exposure.
  • FIGS. 5A and 5B show a time course of RVT-801 dose response in liver and spleen tissue extracts, respectively, indicating that RVT-801 exposure in tissues increased with dose (1, 3, and 10 mg/kg).
  • the highest RVT-801 concentrations were achieved in liver at ⁇ 4 hr post dose and remained elevated beyond the last sampling time point (24 hr) where serum levels were undetectable. Variability between sampling time-points was observed likely owing to complexity of dosing juvenile mice ( ⁇ 16 g bodyweight).
  • pharmacokinetics of RVT-801 were assessed in these two key organs.
  • AUC 0-last increased for liver and spleen at each of efficacious doses.
  • tissue: serum ratio (AUC tissue :AUC serum ) demonstrated markedly higher exposure per organ relative to systemic exposure over a range of efficacious dose.
  • the liver:serum ratios are depicted in FIG. 6B .
  • RVT-801 pharmacokinetics of RVT-801 in these two organs were assessed in comparison with those in a variety of other organs. As shown in FIG. 7 , RVT-801 distributes extensively to tissues associated with ceramide accumulation in the Farber mouse as discussed above in Example 1. Across other tissues, 10 mg/kg RVT-801 achieved tissue:serum ratios as depicted in FIG. 8 .
  • HED Human Equivalent Dose
  • BSA body surface area
  • the 10 mg/kg dose of RVT-801 was scaled by tissue: bodyweight ratios to account for the relative size of each compartment of RVT-801 distribution within each species, using the following formula:
  • HED animal dose ⁇ (Tissue Mass human /BW human )/(Tissue Mass mouse /BW mouse ) (where animal dose is 10 mg/kg).
  • HED human equivalent dose
  • the objective of this study was to determine the serum and tissue pharmacokinetics of RVT-801 following a single dose treatment to juvenile male CD-1 mice of 1, 3, or 10 mg/kg, administered as a bolus IP injection.
  • the maximum effective dose (MED) in Farber mice was 10 mg/kg delivered by bolus IP injection consistent with the current study.
  • the value for AUC last , C max and T max determined following a single 10 mg/kg dose to aged-matched, wild-type CD-1 mice were as reported above:
  • RVT-801-9013A focused on characterizing serum PK:
  • RVT-801-9013B focused on tissue distribution of RVT-801 and tissue:serum exposure ratios
  • the objective of part A of this study was to determine the serum pharmacokinetics of RVT-801 following a single dose to juvenile male CD-1 mice of 1, 3, or 10 mg/kg, administered as a bolus intraperitoneal injection.
  • the objective of part B of the study was to characterize the disposition of RVT-801 in key tissue implicated in ceramide accumulation in the Farber mouse model following a single dose of 1, 3, or 10 mg/kg administered as a bolus intraperitoneal injection to juvenile male CD-1 mice.
  • RVT-801 Drug Substance Source 753-01-16-002 was used in this study.
  • the vehicle formulation used for IP administration was sterile saline.
  • RVT-801 was administered via bolus intraperitoneal injection to male CD-1 mice, aged approximately 3.5 weeks. A total of 108 mice were used in part A of the study and 102 mice were used in part B of the study. NeoSome coordinated animal shipment and supply dates with their supplier (Charles River Laboratories) to allow sufficient acclimation of the pups at the vivarium prior to dosing.
  • Dose material was prepared by diluting the provided stock solution of RVT-801 with sterile PBS according to Table 5.
  • Pharmacokinetic samples were collected on the schedule detailed in Table 7, with whole blood collections from three animals per time point per dose group. The entire volume of whole blood collected from each animal was processed to serum.
  • dose material was prepared by diluting the provided stock solution of RVT-801 with sterile PBS according to Table 8.
  • Blood collections were divided to provide whole blood and serum samples (where indicated) for each animal.
  • the serum PK samples were stored at approximately ⁇ 70° C. and were shipped on dry ice via overnight courier to BioAgilytix (the bioanalytical vendor), where determination of RVT-801 concentrations was performed by sandwich enzyme-linked immunosorbent assay (ELISA).
  • ELISA sandwich enzyme-linked immunosorbent assay
  • Liver and spleen were collected from all dose groups directly following blood collection at the appropriate time point. Tissues were gently blotted dry and placed into pre-labeled (pre-weighed) vials. The weights of each vial containing tissues was recorded, and samples were stored at ⁇ 70° C. until shipment. No buffers, preservative, or antibiotics were added to the tissues, and no tissue perfusions were performed at any stage of the study. For the 10 mg/kg dose group, brain, kidney, heart, and lung were collected and processed as described for liver and spleen.
  • bronchoalveolar lavage fluid was collected from the 10 mg/kg dose group directly following blood collection at the appropriate time point via a pulmonary lavage technique instilling lungs with 0.5 mL phosphate buffered saline and subsequent collection of approximately 0.35 mL of BALF.
  • BALF samples were placed in a suitable container and centrifuged to generate supernatant and an alveolar macrophage pellet. The supernatant was removed and placed into a separate vial. Supernatant and cell pellet were stored at ⁇ 70° C. until shipment.
  • mice Three additional mice were used to provide drug-free matrices.
  • the control matrix samples were used as pre-dose samples for each dose group. Sample collection, processing and storage was performed as described above. Concentrations of RVT-801 in serum were determined using a qualified sandwich ELISA method (BioAgilytix BAL-17-333-036.01-REP). The LLOQ of the serum assay was 20 ng/mL, and the ULOQ of the assay was 1224 ng/mL.
  • the serum method was adapted for quantification in blood, liver, spleen, kidney, heart, lung, and brain.
  • the modified method included preparation of tissue lysates, quantification of total protein using a bicinchoninic acid (BCA) assay and dilution to a total protein concentration of 1 mg/mL in lysis buffer, and preparation of RVT-801 standard curve in lysis buffer. Due to low protein content, BALF samples were processed on a volumetric basis rather than on the basis of standardized protein concentration.
  • the adapted method had a minimum required dilution (MRD) of 50-fold only for mouse serum quality control samples. Standards and study samples were not subjected to the MRD. The signal generated was proportional to the concentration of RVT-801 in the sample.
  • MRD minimum required dilution
  • the LLOQ of the adapted assay was 0.400 ng/mL, and the ULOQ of the assay was 24.5 ng/mL. Measured RVT-801 concentrations were corrected for tissue homogenization and lysate dilution. A description of the bioanalytical method and results are provided in the final bioanalytical report.
  • Pharmacokinetic parameters in Table 10 were derived using noncompartmental methods employing Phoenix WinNonlin® version 7.0 (Certara, Princeton, N.J.) using composite serum and tissue concentration-time data.
  • the area under the concentration-time curve from zero time (predose) to the last non-zero time point was calculated by a combination of linear and logarithmic trapezoidal methods.
  • the linear trapezoidal method was employed for all incremental trapezoids arising from increasing concentrations and the logarithmic trapezoidal method was used for those arising from decreasing concentrations (linear up/log down method). Nominal blood sampling and tissue collection times were used in the analysis.
  • RVT-801 concentration values were received from the bioanalytical lab in units of ng/mL and were converted to units of ⁇ g/mL for PK analysis and reporting. Concentration data and PK parameters were reported to 3 significant figures except for T max and T last values that were reported to 2 decimal places.
  • Mean RVT-801 concentration-time data for the 10 mg/kg dose group are displayed with tissues overlaid on linear axes in FIG. 20 .
  • FIGS. 20A and 20B show Mean RVT-801 Tissue Concentration-Time Profiles Following IP Administration to Juvenile Mice in Part B (Linear and Log-Linear), respectively.
  • RVT-801 concentrations in tissues were quantifiable at the first time point post dose and were highest at 0.5 h post dose for lung and 1 h post dose in liver, spleen, kidney, and heart. Concentrations of RVT-801 generally remained quantifiable in tissues through 18 h post dose.
  • FIG. 21 presents RVT-801 Tissue: Serum Exposure Ratios based on AUC last .
  • a correction factor to account for the dilution is required to reflec concentrations in ELF.
  • FIG. 22A depicts dose normalized area under the curve (DNAUC) data in blood after administration of RVT-801 at 1 mg/kg, 3 mg/kg, and 10 mg/kg.
  • DNAUC dose normalized area under the curve
  • FIG. 22B depicts dose normalized area under the curve (DNAUC) data in serum after administration of RVT-801 at 1 mg/kg, 3 mg/kg, and 10 mg/kg.
  • DNAUC dose normalized area under the curve
  • FIG. 22C depicts dose normalized area under the curve (DNAUC) data in liver tissue after administration of RVT-801 at 1 mg/kg, 3 mg/kg, and 10 mg/kg.
  • DNAUC dose normalized area under the curve
  • FIG. 22D depicts dose normalized area under the curve (DNAUC) data in spleen after administration of RVT-801 at 1 mg/kg, 3 mg/kg, and 10 mg/kg.
  • DNAUC dose normalized area under the curve
  • RVT-801 was widely distributed to tissues, with tissue to serum exposure (AUCiast) ratios ranging from 0.000165 (BALF:Serum) to 92.9 (Liver:Serum). Highest exposure was observed in liver>spleen>kidney>lung>heart>whole blood>BALF with T max ranging from 0.25 h to 2 h post-dose. Exposures in liver, spleen, kidney, heart, and lung were quantifiable through 18 to 24 hours post-dose.
  • AUCiast tissue to serum exposure
  • BALF samples consisted of ELF (epithelial lining fluid) diluted in the phosphate buffered saline (PBS) instillation fluid.
  • ELF epidermal lining fluid
  • PBS phosphate buffered saline
  • the alveolar cellular population was not present in the assayed samples as cells were removed by centrifugation immediately following BALF collection.
  • a correction factor reflecting the dilution of native lung fluid in PBS is required to convert RVT-801 concentrations in BALF to levels in ELF. Determination of the absolute dilution factor was not performed as part of this exploratory study.
  • the correction factor can be determined by comparing the urea content in BALF to the urea content in the plasma from the same animal as native urea levels in epithelial fluid are generally accepted to reflect plasma urea content (Rennard S I, Basset G, Lecossier D, O'Donnell K M, Pinkston P, Martin P G, Crystal R G, 1986, “Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution,” J of Applied Physiology. 1986, 60(2): 532-538).
  • RVT-801 Concentrations of RVT-801 were lower in blood than in serum with blood:serum ratios ranging from 0.127 to 0.584 suggesting RVT-801 does not preferentially associate with erythrocytes. See Table 17.
  • the objective of this study was to determine the serum and tissue pharmacokinetics of RVT-801 following a single dose treatment to juvenile male CD-1 mice of 1, 3, or 10 mg/kg, administered as a bolus IP injection. Following IP administration to juvenile mice, mean serum RVT-801 concentrations were highest between 0.25 and 1 h following dose administration for all dose groups and declined rapidly with an estimated half-life of 1 h. Serum RVT-801 increased with increasing dose, with C max and AUC last increasing greater than proportional with respect to dose. The Vz/F exceeded total body water and the CL/F exceeded liver blood flow. There was a trend towards decreasing Vz/F and CL/F as the dose increased from 3 mg/kg to 10 mg/kg suggesting saturation in tissue distribution and clearance.
  • the maximum effective dose (MED) in Farber mice was 10 mg/kg delivered by bolus IP injection consistent with the current study.
  • the value for AUC last determined following a single 10 mg/kg dose to aged-matched, wild-type CD-1 mice was 1.37 ⁇ g.h/mL with a C max of 1.23 ⁇ g/mL.
  • RVT-801 underwent rapid clearance from systemic circulation consistent with extensive distribution from serum to other tissues. Highest exposures of RVT-801 were observed in liver>spleen>kidney>lung>heart>whole blood>BALF. Tissue to serum exposure ratios based on AUCiast ranged from 0.000165 (BALF:Serum) to 92.9 (Liver: Serum). Tissue exposure persisted generally through 18-24 hours post dose. Concentrations of RVT-801 were lower in blood than in serum with blood:serum ratios ranging from 0.127 to 0.584 suggesting RVT-801 does not preferentially associate with erythrocytes.
  • RVT-801 Male and female Farber mice, aged 4-5 weeks at the time of dose initiation, were administered either single or repeat, once-weekly doses of RVT-801 via bolus intraperitoneal (IP) injection. Dose material was prepared as indicated in Table 19. Individual doses were calculated based on bodyweights recorded on the day of dose administration. RVT-801 was diluted in sterile saline and dose material was prepared fresh on each day of administration.
  • IP intraperitoneal
  • the experiment was designed to determine the concentration of rhAC in circulation and in tissues at selected time points following either single or repeat administration of RVT-801 to Farber mice and to assess the range and extent of RVT-801 distribution to key tissues in relation to the pharmacokinetics established in the CD-1 mouse.
  • the rhAC administered in Examples 5 and 6 comprised a 10 mg/ml (actually 9.91 mg/ml) solution of rhAC at pH 7.4.
  • mice were euthanized in accordance with standard operating procedure and PK samples were collected at 6- and 24-hours post-administration (sample collection followed the third weekly injection of RVT-801 in the repeat dose group).
  • Pharmacokinetic sample collection was restricted to these two time points due to limited available of Farber mice.
  • Whole blood was collected via cardiocentesis into lithium heparin vials and processed in its entirety to plasma. Intact livers, spleens, kidneys, lungs, hearts, brains, and thymic tissue were harvested from each animal, gently blotted dry, and placed into individual vials.
  • Tissues were neither fixed nor perfused with buffer at any stage, and samples were frozen without the addition of buffers, preservatives, protease inhibitors, or antibiotics. Plasma and tissue samples were stored at approximately ⁇ 70° C. and shipped on dry ice to the bioanalytical facilities for analysis. PK sample collection is detailed in Table 20.
  • the concentration of rhAC in Farber mouse tissue following administration of RVT-801 was measured via ELISA conducted by BioAgilytix (Durham, N.C.).
  • Tissue samples were run against calibration standards prepared in homogenization buffer with either serum or buffer-based quality control (QC) samples.
  • QC quality control
  • Incurred tissue samples were homogenized in buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5, 2% (w/v) CHAPS, Thermo Scientific) containing protease inhibitor cocktail (HALT, Thermo Scientific). Samples were processed with a single 5 mm stainless steel bead at 30 Hz using a TissueLyser II (Qiagen) for three cycles of three minutes apiece. Homogenates were placed at 2-8° C. between cycles to control sample temperature.
  • buffer 150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5, 2% (w/v) CHAPS, Thermo Scientific
  • HALT protease inhibitor cocktail
  • the total protein concentration of each tissue homogenate was determined with a colorimetric bicinchoninic acid (BCA) protein quantitation kit (Pierce), and samples were diluted to a nominal total protein concentration of 1 mg/mL with buffer prior to analysis by ELISA.
  • BCA colorimetric bicinchoninic acid
  • Capture antibody (rabbit anti-rhAC) was coated onto a microtiter plate. The plate was blocked with proteinaceous buffer and washed to removed excess capture and blocking reagents. Diluted tissue homogenates were added to the plate along with calibration standards and QCs and incubated to allow conjugation of the rhAC in the samples and controls with the antibody coating the plate. The plate was washed to remove unconjugated rhAC and incubated with the detection antibody (rabbit anti-rhAC-HRP). The plate was washed to remove unbound detection antibody and incubated with the addition of the colorimetric substrate (3,3′,5,5′-tetramethylbenzidine (TMB)) to visualize rhAC conjugated with the detection antibody.
  • TMB colorimetric substrate
  • Sample absorbance was read following the addition of stop solution.
  • the assay generated a signal proportional to the concentration of rhAC in the sample using the parameters outlined in Table 21.
  • RVT-801 concentrations in tissue homogenates were interpolated from the standard curve and corrected by application of dilution factors to calculate the concentration in native tissue.
  • rhAC in Farber mouse plasma was determined by qualified ELISA analysis. Briefly, a microtiter plate was coated with capture reagent (rabbit anti-rhAC), blocked, washed, and incubated with incurred plasma samples and matrix-matched calibration standards and QCs. Following incubation, the plate was washed to remove unbound rhAC and then incubated with the detection reagent (rabbit anti-rhAC-HRP). Excess (unbound) detection reagent was removed by further washing and the colorimetric substrate (3,3′,5,5′-tetramethylbenzidine (TMB)) was added to the plate. Finally, stop solution was added to quench the colorimetric development and the absorbance of the samples was read. The intensity of the colorimetric signal generated in the assay was proportional to the concentration of rhAC in the samples. See Table 22 for a summary of the assay parameters. RVT-801 concentration in Farber mouse plasma samples was back-calculated from the standard curve.
  • RVT-801 Plasma ELISA Summary Sample Matrix Plasma, Lithium Heparin Laboratory Syneos Health RVT-801 Batch 753-01-16-002 Capture Antibody Affinity-purified rabbit anti-rhAC (Thermo Fisher, Lot PTH5119) Detection Antibody Affinity-purified HRP 4 -conjugated rabbit anti-rhAC (Thermo Fisher, Lot 536940-1) MRD 1 50-fold LLOQ 2 20.0 ng/mL ULOQ 3 1280 ng/mL Colorimetric 3,3′,5,5′-tetramethylbenzidine (TMB) Substrate Detection Method Absorbance at 450 (650) nm Plate Reader/Data SpectraMax Plus/SoftMax Pro v5.2 (Molecular Capture Software Devices) Regression Model 4-parameter, 1/y 2 weighting Assay Acceptance Criteria Calibration BCC 5 ⁇ 20% nominal ( ⁇ 25% LLOQ); Standards CV ⁇ 20% ( ⁇ 25% LLOQ
  • RVT-801 concentration data were reported to three significant figures, and T max values were reported to two decimal places.
  • RVT-801 Following repeat administration of RVT-801 at 10 mg/kg/dose, rhAC levels in circulation and across the tissues collected at 6 and 24 hours post-dose were generally lower than those following a single dose of RVT-801 at 10 mg/kg in the CD-1 mice (RVT-801-9021), with multiple 24-hour repeat dose samples below the limit of quantitation. This trend is readily apparent amongst the three tissues with the highest overall rhAC content (liver, spleen, and kidney) following RVT-801 administration to
  • FIG. 21 presents RVT-801 tissue:serum exposure ratios in BALF, blood, brain, heart, kidney, liver, lung, and spleen based on AUClast following single doses of RVT-801 of 1 mg/kg, 3 mg/kg, and 10 mg/kg to juvenile CD-1 mice.
  • Table 23 Mean rhAC concentrations across selected biological matrices for Farber (Asah1 P361R/PP361R ) mice after receiving either a single dose or repeat once-weekly doses of RVT-801 at 10 mg/kg/dose via bolus IP injection.
  • Animals in RVT-801-9025 Part B received four, once-weekly doses of RVT-801, while mice receiving multiple doses in RVT-801-9021 were administered three, once-weekly injections of RVT-801.
  • ELISA Calibrated Range 20.0-1224 ng/mL (serum), 0.400-24.5 ng/mL (tissue), 20.0-1280 ng/mL (plasma).
  • Table 24 presents individual rhAC concentrations across selected biological matrices for Farber (Asah1 P361R/P361R ) mice after receiving either a single dose or repeat once-weekly doses of RVT-801 at 10 mg/kg/dose via bolus IP injection.
  • Animals in RVT-801-9025 Part B received four, once-weekly doses of RVT-801, while mice receiving multiple doses in RVT-801-9021 were administered three, once-weekly injections of RVT-801.
  • ELISA Calibrated Range 20.0-1224 ng/mL (serum), 0.400-24.5 ng/mL (tissue), 20.0-1280 ng/mL (plasma).
  • RVT-801 The murine pharmacokinetics of RVT-801 were characterized previously in healthy, juvenile male CD-1 mice, aged ⁇ 3.5 weeks (RVT-801-9013 Part A). Mice were dosed just after weaning to approximate the size of Farber mice and their age at the initiation of the efficacy studies undertaken in the severe Farber model. The decision to employ the CD-1 mouse for general ADME studies was based on the common genetic background (parental strain of the Farber mouse) along with the cachexic nature and characteristic fragility of the severe Farber mouse influencing their suitability and availability to describe a full concentration-time course of administered RVT-801.
  • RVT-801 When administered as a single IP bolus injection to healthy juvenile male CD-1 mice at 1, 3, or 10 mg/kg, the systemic exposure of RVT-801 generally increased in a supra-proportional manner with respect to dose in terms of both C max and AUC, with concentrations in serum peaking within 1 hour of administration. While relatively short-lived in circulation (half-life ⁇ 1 hour), the apparent volume of distribution and apparent clearance were consistent with rapid and extensive distribution of RVT-801 beyond the vasculature as these values exceeded total body water and hepatic blood flow, respectively (Table 25).
  • FIG. 15 shows final rhAC tissue:serum AUC ratio in healthy juvenile CD-1 mice following a single 10 mg/kg dose of RVT-801.
  • RVT-801 When administered as a single IP bolus injection to juvenile male CD-1 mice at 1, 3, or 10 mg/kg, RVT-801 achieved extensive distribution to a range of selected tissues and organs (RVT-801-9013 Part B). Ranking tissues in order of exposure indicated liver>spleen>kidney>lung>heart>serum>blood, based on AUC ( FIG. 15 , Table 9).
  • RVT-801 did not preferentially associate with erythrocytes. Significantly prolonged durations of exposure in tissue relative to serum were observed; whereas RVT-801 was quantifiable in serum for up to 4-6 hours after dosing, tissue exposure generally persisted for at least 18-24 hours post-administration.
  • RVT-801 Pharmacokinetics in CD-1 and Farber Mice Following a Single 10 mg/kg IP Dose Strain CD-1 Mouse Farber Mouse (Asah1 P361R/P361R ) Matrix Serum Liver Spleen Kidney Lung Heart Plasma Liver Spleen Kidney Lung Heart NCA 1.49 138 70.9 25.8 8.40 4.01 Not Determined 1 AUC last ( ⁇ g ⁇ hr/mL) AUC tissue / — 92.6 47.6 17.3 5.64 2.69 AUC serum C max 2.17 13.0 7.58 2.61 1.42 0.362 0.359 26.2 17.4 2.57 1.64 0.659 ( ⁇ g/mL) T max (hr) 0.25 4.00 4.00 0.25 0.25 0.25 6.00 2 Time Post- Admini- stration (hr) Mean RVT-801 Concentration ( ⁇ g/mL) 0.25 2.17 4.79 5.32 2.61 1.42 0.362 Not Meausred 3 0.5 0.467 5.4 5.74 2.22 1.18
  • FIG. 17 A comparison of rhAC in circulation and in the major tissues of exposure following a single dose of RVT-801 is illustrated in FIG. 17 .
  • FIG. 18 A comparison of the spread of individual data points is presented in FIG. 18 , where the variability of rhAC concentration at a given time point following a single dose of RVT-801 can be appreciated across the tissues analyzed. Again, the increased exposure in Farber relative to CD-1 mice at 24 hours post-dose is readily apparent.
  • FIG. 3 shows that the juvenile CD-1 mouse RVT-801 pharmacokinetic profile is similar to the Farber mouse experimental rhAC activity profile in blood, This indicates that the juvenile CD-1 mouse provides an appropriate approximation of the Farber mouse PK—since Farber mice are frail, difficult to mate, and not numerous enough to conduct a full PK assessment.
  • RVT-801 exposure is likely not the result of analyzing different matrices between strains, as the protein content (aside from clotting factors) of serum and plasma is largely similar. Additionally, the increase in measured Farber plasma exposure was not a result of increased plasma ELISA sensitivity relative to that in serum and/or the ability to report additional data points (i.e. at 24 hours post-dose) that would have been undetectable in CD-1 serum due to differences in method sensitivity. Reported Farber plasma data were well above the LLOQ and the plasma and serum methods performed similarly (Table 27).
  • FIGS. 19A-G present the mean rhAC concentration in CD-1 mice following a single 10 mg/kg dose and Farber mice following either a single 10 mg/kg dose or multiple once-weekly doses at a 10 mg/kg/dose administered via bolus IP injection, in accordance with Example 5 (RVT-801-9021).
  • FIGS. 20A and 20B show mean rhAC tissue concentration-time profiles following IP administration to juvenile CD-1 mice in RVT-801-9013 Part B (Linear FIG. 20A and Log-Linear FIG. 20B ), respectively.
  • FIG. 21 presents RVT-801 tissue:serum exposure ratios in BALF, blood, brain, heart, kidney, liver, lung and spleen based on AUC last based on single doses of RVT-801 of 1 mg/kg, 3 mg/kg and 10 mg/kg in CD-1 mice.
  • FIGS. 22A-D presents dose normalized PK parameters plotted verses dose level for various tissues following IP administration of RVT-801 at doses of 1 mg/kg, 3 mg/kg and 10 mg/kg in CD-1 mice.
  • FIG. 19A presents a plot of systemic rhAC concentrations in serum after a single dose administration of 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19A also presents plasma concentrations in Farber mice after dose administration of 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19A presents plasma concentrations following a repeat dose administration of injection of RVT-80.
  • FIG. 19B presents concentrations of RVT-801 in liver tissue in CD-1 mice after a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19B also presents concentrations of RVT-801 in liver tissue in Farber mice treated with a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19B additionally presents concentrations of RVT-801 in liver tissue in Farber mice treated with repeated 10 mg/kg/doses via bolus IP injection of RVT-801.
  • FIG. 19C presents concentrations of RVT-801 in spleen tissue in CD-1 mice after a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19C also presents concentrations of RVT-801 in spleen tissue in Farber mice treated with a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19C additionally presents concentrations of RVT-801 in liver tissue in Farber mice treated with repeated 10 mg/kg/doses via bolus IP injection of RVT-801.
  • FIG. 19D presents concentrations of RVT-801 in kidney tissue in CD-1 mice after a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19D also presents concentrations of RVT-801 in kidney tissue in Farber mice treated with a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19C additionally presents concentrations of RVT-801 in kidney tissue in Farber mice treated with repeated 10 mg/kg/doses via bolus IP injection of RVT-801.
  • FIG. 19E presents concentrations of RVT-801 in heart tissue in CD-1 mice after a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19E also presents concentrations of RVT-801 in heart tissue in Farber mice treated with a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19E additionally presents concentrations of RVT-801 in heart tissue in Farber mice treated with repeated 10 mg/kg/doses via bolus IP injection of RVT-801.
  • FIG. 19F presents concentrations of RVT-801 in lung tissue in CD-1 mice after a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19F also presents concentrations of RVT-801 in lung tissue in Farber mice treated with a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19F additionally presents concentrations of RVT-801 in lung tissue in Farber mice treated with repeated 10 mg/kg/doses via bolus IP injection of RVT-801.
  • FIG. 19G presents concentrations of RVT-801 in brain tissue in CD-1 mice after a single 10 mg/kg/dose via bolus IP injection of RVT-801.
  • FIG. 19G additionally presents concentrations of RVT-801 in brain tissue in Farber mice treated with repeated 10 mg/kg/doses via bolus IP injection of RVT-801.
  • ASAH1 acid ceramidase gene.
  • AUC area under the concentration-time curve.
  • AUC (0- ⁇ ) area under the plasma concentration-time curve from time zero extrapolated to time infinity.
  • AUC (0-t) area under the plasma concentration-time curve from time zero to last sample time.
  • BMI body mass index
  • cDNA complementary deoxyribonucleic acid
  • CNS central nervous system
  • HED human-equivalent dose
  • HSCT hematopoietic stem cell transplantation.
  • IP intraperitoneal.
  • IV intravenous
  • MCP-1 monocyte chemoattractant protein 1.
  • PK pharmacokinetic(s).
  • PRN pro re nata.
  • rhAC recombinant human acid ceramidase
  • SMA-PME Spinal Muscular Atrophy with Progressive Myoclonic Epilepsy.
  • Food and Drug Administration 2005, July, “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers.” Available at: https://www.fda.gov/downloads/drugs/guidances/ucm078932.pdf.
  • Food and Drug Administration 2015, May, “Draft Guidance: Investigational Enzyme Replacement Therapy Products: Nonclinical Assessment.” Available at: https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guid ances/UCM446569.pdf.
  • Torcoletti M Petaccia A, Pinto R M, Hladnik U, Locatelli F, Agostoni C, Corona F., 2014, “Farber disease in infancy resembling juvenile idiopathic arthritis: identification of two new mutations and a good early response to allogeneic haematopoietic stem cell transplantation. Rheumatology (Oxford), August; 53(8):1533-4.
  • the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term about generally refers to a range of numerical values (e.g., +/ ⁇ 5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • the terms modify all of the values or ranges provided in the list.
  • the term about may include numerical values that are rounded to the nearest significant figure.

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